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<div class='MSDStitle'>Materials</div>
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				<li><span style='font-weight:bold;'>HDFf Medium:</span> DMEM (10%FBS, 1% Non-essential amino acid)</li>
				<li><span style='font-weight:bold;'>iPSC Medium:</span> DMEM (20% knock-out serum replacer, 200mM  L-glutamine, 1% Non-essential amino acid, 8ng/ml b-FGF )</li>
				<li><span style='font-weight:bold;'>Recombinant iPSC adenoviral vectors (either set or polycistronic vectors):</span>All recombinant adenoviral vectors purchased from ABM are purified high titer preparation and ready to use.</li>
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	  <div class='MSDStitle'>Protocol</div>
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          	<td colspan=2 valign='top'><span style='font-weight:bold;'>The following protocol is based on human fibroblasts and can be applied to most other cell types:</span></td>
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				<li>Seed HDF cells in 10cm dishes (20-30% confluent) one day before viral infection, including one dish used for GFP control.</li>
				<li>The following day, dilute 50uL of each adenoviral vector (individual ones or polycistronic one) into 5mL complete culture medium.</li>
				<li>Aspirate medium from 10cm dishes and add diluted virus (5mL) to cells. Return dishes to a CO2 incubator for an hour. </li>
				<li>Aspirate virus-containing medium, and replenish with 10mL iPSC medium.</li>
				<li>Change media every 48 hours.</li>
				<li>Repeat adenoviral gene transduction every 5 days as transgene expression by adenoviral vector will only last 5~7 days, depending on the rate of cell division.</li>
				<li>Wait 2-4 weeks for iPSC colonies to form.</li>
				<li>Once iPSC colonies formed in 10cm dishes. Prepare Mytomycin C treated MEF (mouse embryonic fibroblasts) feeder cells in 24-well plate (80% confluent) at a concentration of 10mg/mL for 3 hours in a incubator at 37C followed by 2x PBS wash.</li>
				<li>Pick colonies manually into 96-well plate and trypsinize in 96-well plate.</li>
				<li>Transfer the trypsinized cells from each of the 96 wells into 24-well MEF coated plate.</li>
				<li>Wait for another 1-2 weeks for iPSC colonies to develop (change medium every 48 hours).</li>
				<li>When MEF cells become too old (about 2 weeks) or a lot of iPSC colonies developed in the 24-well plate, prepare MEF feeder layer as described above in 6-well plate to expand.</li>
				<li>Trypsinize the cells (both MEF and iPSC cells) and spin down at 1000xg.</li>
				<li>Then, seed iPSCs into 6-well MEF coated plate for expansion.</li>
				<li>Using the same procedures above, iPSCs can be further expanded to bigger culture vessels to meet your lab needs for iPSC analysis or down-stream applications.</li>
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